Transcriptome and proteome analyses reveal genes and signaling pathways involved in the response to two insect hormones in the insect-fungal pathogen Hirsutella satumaensis

Abstract

The insect hormones ecdysone (20E) and juvenile hormone III (JH) have been demonstrated to stimulate the secretion of conidia mucilage and pigments in Hirsutella satumaensis. However, the underlying mechanisms remain elusive. Here, comparative transcriptome and proteome analyses were performed to identify the fungal genes and proteins of H. satumaensis that are up- or downregulated in response to insect hormones. A total of 17,407 unigenes and 1,016 proteins in conidia mucilage were identified. The genes involved in response to the hormones were classified into four functional groups: (1) stress response-related genes that are required for the removal of reactive oxygen species (glutathione synthetase, c7144) and genes involved in the response to osmotic stress in the hemocoel, such as those encoding proteins involved in the G, mTOR, and MAPK signaling pathways (2); insect hormone metabolic genes, including genes encoding ecdysteroid UDP-glucosyltransferase, ecdysteroid-22-kinase, and a key aldehyde dehydrogenase in a juvenile hormone synthesis pathway (3); secretory proteins that share homology with those of the host Bombyx mori, including fibrohexamerin, sericin 1, metalloprotease 1 protein, and silk gum protein, which were revealed by the omics data; and (4) proteins related to amino sugar metabolism and oxidative phosphorylation that were specifically expressed in mucilage in response to 20E and JH, respectively. These findings revealed that H. satumaensis can mount effective responses by modulating the expression of genes involved in the detoxification, adaptation, and evasion of insect hormone-mediated immune responses, providing fresh insights into fungal pathogen-host insect interactions.IMPORTANCEInsect hormones are highly important for the regulation of insect growth, development, and immune system function. Thus, the expansion of entomopathogenic fungi (EPF) could be affected by these hormones when they inhabit the host hemocoel. However, the molecular basis of EPF in response to insect hormones has yet to be determined. Our results revealed that EPF are impacted by 20E and JH, both of which act as signals, as these hormones lead to changes in metabolic pathways of the fungus, thus demonstrating a direct relationship between the fungus and the hormones. Furthermore, adaptive strategies, such as the use of ecdysone-inactivating enzymes and secreted filamentous proteins in H. satumaensis, which strongly resemble those of the host insect, have been discovered, thus illustrating the importance of adaptation to insect hormones for a better understanding of the interaction between insects and EPF.

Keywords: Hirsutella satumaensis; conidia mucilage; ecdysone; juvenile hormone III; transcriptome and proteome.

Fig 1

Differentially expressed genes (DEGs) analysis. (A) Number of upregulated and downregulated DEGs associated with the two insect hormones under the induced status in H. satumaensis (20E: ecdysone, JH: juvenile hormone III; same below). (B and C) Volcanograms of DEGs under 20E and JH pressure, respectively. (D and E) KEGG enrichment analysis of significantly up and downregulated unigenes of H. satumaensis under 20E and JH stress, respectively.

Fig 2

The functional classification of significantly up- and downregulated DEGs of H. satumaensis under the stress of 20E and JH, respectively. The abbreviations for the functional classification are CSF (cell structure and function), SRD (stress response and defense), CCNM (cell cycle and nucleic acid metabolism), CM (carbohydrate metabolism), LM (lipid metabolism), EN (energy metabolism), SM (secondary metabolism), NPM (nitrogen and protein metabolism), SITG (signal transduction and gene transcription), UN (unknown), and PHI (pathogen-host interactions).

Fig 3

The enrichment score of the metabolic pathway and the top genes expressed in the 20E and JH groups were analyzed by GSEA based on the RNA-seq data sets. The 20E treatment group showed significant enrichment in the tyrosine pathway (A), while the ribosome pathway was notably enriched in the JH treatment group (C). The top 8 and 15 most highly expressed genes in the 20E (B) and JH (D) groups, respectively. NES, a normalized enrichment score. P values in the graphs were calculated by GSEA analysis.

Fig 4

Classification and analysis of secretory proteins from the transcriptome data of H. satumaensis under 20E and JH induction conditions. (A) Functional classification of the co-expressed secretory proteins. (B) The Venn diagram shows the number of identified proteins that are significantly up- and downregulated. The functional classification abbreviations are CTM (carbohydrate transport and metabolism), LTM (lipid transport and metabolism), ITM (inorganic ion transport and metabolism), CWMEB (cell wall/membrane/envelope biogenesis), PMPTC (post-translational modification, protein turnover, chaperones), ITSVT (intracellular trafficking, secretion and vesicular transport), GFP (general function prediction only), and UN (unknown).

Fig 5

Analysis of differentially expressed proteins (DEPs). (A) Number of DEPs associated with mucilage induced by the two insect hormones. (B and C) Hierarchical clustering of specifically expressed proteins in the 20E and JH treatment groups, respectively. (D and E) GO classes of DEPs associated with the mucilage of H. satumaensis under 20E and JH induced, respectively.

Fig 6

Candidates were divided into nine groups according to the log2 ratios of the protein species (x-axis) and transcripts (y-axis). Quadrants 1, 2, and 4 indicate that the protein abundance was lower than the RNA abundance. In quadrants 3 and 7, the RNAs correspond with the related proteins. Quadrant 5 shows that the proteins and RNAs were commonly expressed with no difference. Quadrants 6, 8, and 9 indicate that the protein abundance was higher than the RNA abundance. Comparison of changes in mRNAs and protein abundance of conidial mucilage.

Fig 7

Validation of transcript/protein expression changes under 20E and JH induction conditions by qRT-qPCR. "**" and "*" indicate that the expression levels of two strains are significantly different at P < 0.01 and P < 0.05, respectively. (A) The representative secreted proteins that are co-upregulated in both groups. (B) The representative secreted proteins that are co-downregulated in the two hormone groups. (C) The seven representative proteins that are exclusively secreted upon JH induction. (D) The ribosomal metabolism-related genes. (E) The genes related to hormone metabolization.